Compositions and methods for treating and preventing Staphylococcus aureus infections

ABSTRACT

Antibodies having Fab regions that specifically bind to  Staphylococcus aureus  protein A are capable of mediating opsinization of  Staphylococcus aureus  bacteria despite their expression of antibody-neutralizing protein A. These antibodies and antigen-binding fragments thereof can be used in methods of treating and/or preventing  Staphylococcus aureus  infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional patentapplication No. 62/007,242 filed on Jun. 3, 2014, U.S. provisionalpatent application No. 62/041,423 filed on Aug. 25, 2014, and U.S.provisional patent application No. 62/115,665 filed on Feb. 13, 2015.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 28, 2015, isnamed 5407-0233_SL.txt and is 83,034 bytes in size.

FIELD OF INVENTION

The invention relates generally to methods of medical treatment,immunology, and microbiology. More particularly, the invention relatesto compositions and methods for treating and preventing Staphylococcusaureus infections.

BACKGROUND

Staphylococcus aureus (SA) is a substantial cause of sickness and deathin both humans and animals. Infection with these gram-positive coccioften results in the development of a superficial abscess. Other casesof SA infection can be much more serious. For example, intrusion of SAinto the lymphatics and blood can lead to a systemic infection which inturn can cause complications such as endocarditis, arthritis,osteomyelitis, pneumonia, septic shock and even death. Hospital-acquiredSA infection is common and particularly problematic with SA being themost frequent cause of hospital-acquired surgical site infections andpneumonia, and the second most frequent cause of cardiovascular andbloodstream infections. Antibiotic administration has been and remainsthe standard treatment for SA infections. Unfortunately, the use ofantibiotics has also fueled the development of antibiotic resistance inSA. Notably, methicillin-resistant SA (MRSA) has evolved the ability toresist beta-lactam antibiotics such as penicillin and cephalosporins.More alarmingly, SA resistant to antibiotics of last resort such asvancomycin and linezolid have recently emerged. Therefore a new approachfor preventing and treating SA infections is needed

SUMMARY

It was discovered that certain antibodies (Abs) having Fab regionparatopes that specifically bind to SA protein A (SpA) are capable ofmediating opsinization of SA bacteria despite SA's expression ofantibody (Ab)-neutralizing SpA. Previous Ab-based strategies fortreating or preventing SA infections showed promise in pre-clinical andearly stage clinical trials, but failed to meet endpoints in phase IIItrials. Perhaps explaining these results, previous strategies did notaddress the Ab-neutralizing property of SpA. SpA is a heavily expressedcell wall-associated protein that binds most immunoglobulins (Igs) viatheir Fc (effector) regions. SpA binds to human antibodies of subclassesIgG1, IgG2, and IgG4 via their Fc region with a KD of about 1×10⁻⁹ M,and thereby acts as an Fc region anchor that orients the effectorportion of an immunoglobulin (Ig) away from Fc-interacting immuneeffectors such as complement and Fc receptor (FcR)-bearing phagocytes.Accordingly, most Abs specific for SA antigens are “sequestered” fromimmune effectors in this manner. In addition, because SpA is so highlyexpressed on the cell wall of SA (estimated 7% of the cell wall), itmediates the formation of a shield of Igs covering the cell wall. Thisshield sterically hinders Abs specific for cell wall antigens frombinding their targets and mediating oponophagocytosis of the bacteria.The formation of an Ig shield was not previously appreciated as avirulence factor. Thus the discovery that SA-binding Abs having Fabregions that specifically bind SpA while permitting their Fc regions tostill interact with FcRs on immune effector cells and/or activatecomplement by binding C1q despite the Fc-neutralizing ability of SpA andthe formation of an Ig shield was a significant step over other anti-SAAb-based approaches. Preferred versions of such Abs are capable ofdisplacing Igs already bound to SpA by their Fc regions.

As examples of the foregoing, described herein are isolated or purifiedantibodies (particularly human IgG3 antibodies which have Fc regionswith low or no affinity for SpA such as one with the allotype havingarginine at amino acid position 435; Stapleton et al., NatureCommunications 2, Article number: 599, 2011) having Fab regions that canspecifically bind a target epitope of SpA on a SA bacterium while theirFc regions are still able to interact with an FcR (e.g., solublerecombinant or native on immune effector cells)—despite the Fc-bindingproperty of SpA and steric hindrance of the target epitope by Igs boundto SpA via their Fc region. Also provided herein are pharmaceuticalcompositions that contain at least one of these antibodies and apharmaceutically acceptable carrier (e.g., a non-naturalpharmaceutically acceptable carrier). Further provided are methods oftreating a subject having a SA infection or reducing the risk ofdeveloping a SA infection in a subject that include administering atherapeutically effective amount of any of the pharmaceuticalcompositions described herein or any of the antibodies orantigen-binding fragments described herein to a subject in need thereof.

As used herein, the word “a” or “an” before a noun represents one ormore of the particular noun. For example, the phrase “an antibody”represents “one or more antibodies.”

By the term “antibody” or “Ab” is meant any immunoglobulin (e.g., human,cartilagenous fish, or camelid antibodies) or conjugate thereof, thatspecifically binds to an antigen (e.g., an SpA antigen such as SEQ IDNO: 1 or an antigenic fragment of SEQ ID NO: 1). A wide variety of Absare known by those skilled in the art. Non-limiting examples of Absinclude: monoclonal Abs (e.g., including full-length Abs), polyclonalAbs, multi-specific Abs (e.g., bi-specific Abs), dual variable domainAbs, single-chain Abs (e.g., single-domain Abs, camelid Abs, andcartilagenous fish Abs), chimeric (e.g., humanized, such as humanizedIgG3) Abs, and human Abs (e.g., human IgG3 Abs). The term antibody alsoincludes Ab conjugates (e.g., an Ab conjugated to a stabilizing protein,a label, or a therapeutic agent (e.g., any of the therapeutic agentsdescribed herein or known in the art)).

By the term “antigen-binding fragment” is meant any portion of afull-length Ab that contains at least one variable domain ((e.g., avariable domain of a mammalian (e.g., human, mouse, rat, rabbit, orgoat) heavy or light chain immunoglobulin), a camelid variableantigen-binding domain (VHH), or a cartilagenous fish immunoglobulin newantigen receptor (Ig-NAR) domain) that is capable of specificallybinding to an antigen. For example, an antigen-binding fragmentdescribed herein can include at least part of an Ab Fc region that issufficient to mediate antibody-dependent cell-mediated cytotoxicity(ADCC) and/or complement-dependent cytotoxicity (CDC) in a mammal (e.g.,a human) and/or is conjugated to a therapeutic agent (e.g., any of thetherapeutic agents described herein or known in the art). Non-limitingexamples of Ab fragments include Fab, Fab′, F(ab′)₂, Fv fragments,diabodies, linear antibodies, and multi-specific Ab formed from Abfragments. Additional Ab fragments containing at least one camelid VHHdomain or at least one cartilagenous fish Ig-NAR domain includemini-bodies, micro-antibodies, subnano-antibodies, and nano-antibodies,and any of the other forms of Abs described in U.S. Patent ApplicationPublication No. 2010/0092470. An antigen binding fragment can be, e.g.,an antigen-binding fragment of human or humanized IgG1, IgG2, IgG3 IgG4,IgD, IgA, IgE, or IgM.

By the term “human antibody” is meant an Ab that is encoded by a nucleicacid (e.g., rearranged human immunoglobulin heavy or light chain locus)present in the genome of a human. In some embodiments, a human Ab isproduced in a mammalian (e.g., human) cell culture. In some embodiments,a human Ab is produced in a non-human cell (e.g., a Chinese hamsterovary cell line or a mouse or hamster cell line). In some embodiments, ahuman Ab is produced in a bacterial or yeast cell. A human Ab caninclude a conjugated therapeutic agent (e.g., any of the therapeuticagents described herein or known in the art). A human Ab can be humanIgG1, IgG2, IgG4, IgD, IgA, IgE, or IgM, and is preferably human IgG3.By the term “true human antibody” is meant an Ab with heavy and lightchain variable regions that are naturally present in the serum of ahuman being.

By the term “humanized antibody” is meant an Ab which contains mostlysequences of a human Ab but also includes minimal sequences derived froma non-human (e.g., mouse, rat, rabbit, or goat) Ig. In non-limitingexamples, humanized Abs are human Abs (recipient Ab) in whichhypervariable region residues of the recipient Ab are replaced byhypervariable region residues from a non-human species Ab (donor Ab),e.g., mouse, rat, rabbit, or goat Ab having the desired specificity,affinity, and capacity. In some embodiments, the Fv framework residuesof the human Ig are replaced by corresponding non-human residues. Insome embodiments, humanized Abs may contain residues which are not foundin the recipient Ab or in the donor Ab. These modifications can be madeto further refine Ab performance.

In some embodiments, the humanized Ab will contain substantially all ofat least one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops (complementary determiningregions) correspond to those of a non-human immunoglobulin and all orsubstantially all of the framework regions are those of a humanimmunoglobulin sequence. The humanized antibody can also contain atleast a portion of an Ig constant region (Fc region), typically, that ofa human Ig (e.g., human IgG3). Humanized Abs can be produced bymolecular biology methods that are well known in the art. Non-limitingexamples of methods for generating humanized Abs are described herein. Ahumanized antibody can include a conjugated therapeutic agent (e.g., anyof the therapeutic agents described herein or known in the art).

By the term “single-chain antibody” is meant a single polypeptide thatcontains at least one variable binding domain (e.g., a variable domainof a mammalian heavy or light chain Ig, a camelid variableantigen-binding domain (VHH), or a cartilagenous fish (e.g., shark)immunoglobulin new antigen receptor (Ig-NAR) domain) that is capable ofspecifically binding to an antigen. Non-limiting examples ofsingle-chain Abs are described herein, and are known in the art (see,for example, the antibodies described in U.S. Patent Publication No.2010/0092470). A single-domain antibody can include a conjugatedtherapeutic agent (e.g., any of the therapeutic agents described hereinor known in the art).

An Ab or antigen-binding fragment thereof “specifically binds” or “bindsspecifically” to a particular antigen, e.g., SpA (such as an epitopecomprising SEQ ID NO: 1 or an antigenic fragment of SEQ ID NO: 1), whenit binds to that antigen, but recognizes and binds to a lesser extent(e.g., does not recognize and bind) to other molecules in a sample. Insome embodiments, an Ab or an antigen-binding fragment thereofselectively binds to an epitope with an affinity (K_(D)) equal to orless than 1×10⁻¹⁰ M (e.g., less than 1×10⁻¹¹ M or less than 1×10⁻¹² M)in phosphate buffered saline (e.g., as determined by surface plasmonresonance). The ability of an Ab or antigen-binding fragment tospecifically bind a protein epitope may be determined using any of themethods known in the art or those methods described herein.

By the term “complementarity determining region” or “CDR” is meant aregion within an Ig (heavy or light chain Ig) that forms part of anantigen-binding site (paratope) in an Ab or antigen-binding fragmentthereof. As is known in the art, a heavy chain Ig normally containsthree CDRs: CDR1, CDR2, and CDR3, respectively, and a light chain Ignormally contains three CDRs: CDR1, CDR2, and CDR3. In any Ab orantigen-binding fragment thereof, the three CDRs from the heavy chain Igand the three CDRs from the light chain Ig together form anantigen-binding site in the Ab or antigen-binding fragment thereof. TheKabat Database is one system used in the art to number CDR sequencespresent in a light chain Ig or a heavy chain Ig.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of SpA showing the different domains andthe location of each of five antigenic peptides. The sequence ofantigenic peptide #5 is shown (SEQ ID NO: 1).

FIG. 2 is a set of two graphs showing a histogram of the fluorescence ofSA clinical isolate 00X (top) and SA strain ATCC #25923 (bottom)incubated with biotinylated PA8-G3 Ab (light line) or controlbiotinylated anti-interleukin-1 alpha Ab (MABp1) (dark line), and thenincubated with streptavidin-APC.

FIG. 3 is a set of two graphs showing a histogram of the fluorescence ofclinical isolate 00X (top) and strain ATCC #25923 (bottom) incubatedwith unlabeled PA8-G3 Ab (light line) or unlabeled MABp1 Ab (dark line),followed by biotinylated recombinant Fcγ receptor 1, and then incubatedwith streptavidin-APC.

FIG. 4 is graph of the mean fluorescent intensity of differentiated HL60cells (using fluorescence cell sorting) following co-incubation withPA8-G3 Ab opsonized with pH-rodo-green labeled strain ATCC #25923 orclinical isolate 00X. Similar samples incubated with a control Ab MABp1,instead of PA8-G3 Ab were used as a negative control.

FIG. 5 is a set of two graphs showing the fluorescence intensity ofclinical isolate 00X (top) or ATCC #25923 (bottom) pre-incubated withhuman sera for 15 minutes prior to the addition of biotinylated PA8-G3Ab or negative control MABP1 Ab, and then incubated with streptavidinAPC.

FIG. 6 is a graph showing the mean fluorescent intensity ofdifferentiated or undifferentiated HL-60 cells after co-incubation withpH-rodo-green labeled SA and one of the following unlabeled Abs:PA7.2-G3, PA4-G3, PA8-G3, PA15-G3, PA21-G3, PA27-G3, PA32-G3, PA37-G3,or MABp1. The MABp1 Ab samples were used as a negative control.

FIG. 7A-D are graphs showing that administration of mAb PA8 enhances thesurvival of murine subjects infected with S. aureus.

FIGS. 8 A-C are graphs showing the synergy between PA8-G3 andvancomycin.

DETAILED DESCRIPTION

Described herein are methods and compositions for treating a subjecthaving a SA infection or reducing the risk of developing a SA infectionin a subject.

Antibodies and Antigen-Binding Fragments Thereof

Described herein are purified or isolated (e.g., at least 90%, 92%, 94%,95%, 96%, 97%, 98%, or 99% pure by weight) Abs (e.g., preferably truehuman, human, or humanized IgG3s) that bind to SpA and are capable ofmediating opsinization of SA bacteria despite SA's expression ofantibody (Ab)-neutralizing SpA. Preferred such Abs bind to the peptideof SEQ ID NO:1 with a sufficient binding affinity to displace human IgGimmunoglobulins (e.g., one or more of IgG1, IgG2, and IgG4) bound to SpAvia their Fc region. Preferred Abs can bind to SpA via their Fab regionparatopes with a K_(D) of less than 1×10⁻¹⁰ M (e.g., less than 1×10⁻¹¹M, less than 1×10⁻¹² M, less than 0.5×10⁻¹² M, or less than 1×10⁻¹³M)under physiological conditions (e.g., phosphate buffered saline) (e.g.,as determined using surface plasmon resonance or Bio-LayerInterferometry using recombinant SpA). For example, the Abs describedherein that bind to SpA via their Fab regions with a K_(D) of between1×10⁻¹⁰ M and 0.5×10⁻¹²M, between 1×10⁻¹¹ M and 0.5×10⁻¹² M, between1×10⁻¹¹ M and 0.2×10⁻¹² M (e.g., under physiological conditions, e.g.,phosphate buffered saline, e.g., as measured used surface plasmonresonance using recombinant SpA) are preferred. Those Abs orantigen-binding fragments described herein preferably are able todisplace human Abs (e.g., one or more of IgG1, IgG2, and IgG4) bound toSpA in the cell wall of a SA bacterium via their Fc regions. Alsoprovided herein are purified or isolated (e.g., at least 90%, 92%, 94%,95%, 96%, 97%, 98%, or 99% pure by weight) mAbs (e.g., preferably truehuman, human, or humanized IgG3s) that specifically bind Staphylococcusaureus protein A (SpA) with a K_(D) of less than 1×10⁻¹⁰ M via their Fabregion paratopes, wherein the mAbs are able to mediate opsinization ofSpA-expressing Staphylococcus aureus bacteria in the presence of atleast 1 mg/ml (e.g., at least 1, 2, 3, 4, 5, 10, 25, 50, or 100 mg/ml,or the amount normally contained in human serum) of IgG immunoglobulinswhich bind SpA via their Fc regions

The purified or isolated Abs provided herein might bind to an epitopepresent in the extracellular domain (e.g., present in the X_(R) repeatregion and one or more of the IgG binding domains) of SpA. Non-limitingexamples of an antigen that can be specifically recognized by any of theAbs (or antigen-binding fragments thereof) provided herein include: 6,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous aminoacids of SEQ ID NO: 1 (e.g., a fragment starting at amino acid position1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of SEQ ID NO: 1); 6, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous aminoacids of SEQ ID NO: 82 (e.g., a fragment starting at amino acid position1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of SEQ ID NO: 82);6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous amino acids of SEQID NO: 83 (e.g., a fragment starting at amino acid position 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 of SEQ ID NO: 83); 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or 21 contiguous amino acids of SEQ ID NO: 84(e.g., a fragment starting at amino acid position 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15 of SEQ ID NO: 84); 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ IDNO: 85 (e.g., a fragment starting at amino acid position 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 of SEQ ID NO: 85); or 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids fromamino acid positions 1 to 20, 10 to 30, 20 to 40, 30 to 50, 40 to 60, 50to 70, 60 to 80, 70 to 90, 80 to 100, 90 to 110, 100 to 120, 110 to 130,120 to 140, 130 to 150, 140 to 160, 150 to 170, 160 to 180, 170 to 190,180 to 200, 190 to 210, 200 to 220, 210 to 230, 220 to 240, 230 to 250,240 to 260, 250 to 270, 260 to 280, 270 to 290, 280 to 300, 290 to 310,300 to 320, 310 to 330, 320 to 340, 330 to 350, 340 to 360, 350 to 370,360 to 380, 370 to 390, 380 to 400, 390 to 410, 400 to 420, 410 to 430,420 to 440, or 430 or to 450 of SEQ ID NO: 86. Examples of otherantigens include similar fragments of SpAs having amino acids sequencesdiffering from that of SEQ ID NO:86.

Methods for determining the ability of an Ab or antigen-binding fragmentthereof to bind to a target protein (e.g., SpA or a portion thereof) canbe performed using methods known in the art. Non-limiting examples ofsuch methods include competitive binding assays using Abs known to bindthe target protein (e.g., SpA), enzyme-linked immunosorbent assays,BioCoRE®, affinity columns, immunoblotting, or protein array technology.In some embodiments, the binding activity of the Ab or antigen-bindingfragment thereof is determined by contacting a SA bacterium with the Abor antigen-binding fragment thereof. Exemplary methods for determiningthe ability of an Ab or antigen-binding fragment to displace human Abs(e.g., one or more of IgG1, IgG2, and IgG4) bound to SpA in the cellwall of a SA bacterium are described in the Examples section below.Additional methods for determining the ability of an Ab orantigen-binding fragment to displace human Abs (e.g., one or more ofIgG1, IgG2, and IgG4) bound to SpA in the cell wall of a SA bacteriumare known in the art.

An Ab can be, e.g., a mAb, a multi-specific Ab (e.g., a bispecific Ab),a chimeric Ab (e.g., a humanized Ab, such as a humanized IgG Ab), ahuman Ab, or a fragment of any of the foregoing. For example, an Ab canbe a human or humanized monoclonal IgG3 Ab. An Ab can also be asingle-chain Ab (e.g., a single-domain Ab), such as a single-chaincamelid or cartilagenous fish (e.g., shark) Ab, or a single-chain Abthat contains at least one camelid variable antigen-binding domain (VHH)or at least one cartilagenous fish (e.g., shark) immunoglobulin newantigen receptor (Ig-NAR) domain (see, for example, the Abs described inU.S. Patent Publication No. 2010/0092470). An Ab can be a whole Abmolecule or an Ab multimer.

The term Ab also includes Ab conjugates (e.g., an Ab conjugated to astabilizing protein, a label, or a therapeutic agent (e.g., any of thetherapeutic agents described herein or known in the art)). An Abprovided herein can, for example, include a Fc domain or part of a Fcdomain that is sufficient to mediate Ab-dependent cell-mediatedcytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) in amammal (e.g., a human), and/or is conjugated to a therapeutic agent(e.g., any of the therapeutic agents described herein or known in theart). An Ab can be, e.g., a human or humanized IgG1, IgG2, IgG4, IgD,IgA, IgE, or IgM, and is preferably a human or humanized IgG3.

An antigen-binding fragment described herein can, e.g., include at leastpart of a Fc domain that is sufficient to mediate Ab-dependentcell-mediated cytotoxicity (ADCC) and/or complement-dependentcytotoxicity (CDC) in a mammal (e.g., a human) and/or is conjugated to atherapeutic agent (e.g., any of the therapeutic agents described hereinor known in the art). Non-limiting examples of Ab fragments include Fab,Fab′, F(ab′)₂, single-chain Fvs (scFvs), Fv fragments, fragmentscontaining either a variable light or variable heavy chain domain,diabodies, linear Abs, and multi-specific Abs formed from Ab fragments.Additional Ab fragments containing at least one camelid VHH domain or atleast one cartilagenous fish Ig-NAR domain include mini-bodies,micro-Abs, subnano-Abs, and nano-Abs, and any of the other forms of Absdescribed in U.S. Patent Application Publication No. 2010/0092470.

The Abs or antigen-binding fragments thereof can be of any type (e.g.,human or humanized IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., humanor humanized IgG1 (e.g., IgG1a or IgG1b), IgG2 (e.g., IgG2a or IgG2b),IgG3 (e.g., IgG3a or IgG3b), IgG4 (e.g., IgG4a or IgG4b), IgA1, and IgA2or subclass, although those with an Fc binding affinity for SpA is low(e.g., having a K_(D) of greater than 1×10⁻⁷ M, 1×10⁻⁶ M, 1×10⁻⁵M,1×10⁻⁴ M, or 1×10⁻³ M; or having a K_(D) greater than that of SpA forthe Fc region of a human IgG1) under physiological conditions (e.g.,phosphate buffered saline) (e.g., as determined using surface plasmonresonance using recombinant SpA) are preferred. An antigen bindingfragment can be, e.g., an antigen-binding fragment of human or humanizedIgG1 (e.g., IgG1a or IgG1b), IgG2 (e.g., IgG2a or IgG2b), IgG4 (e.g.,IgG4a or IgG4b), IgD, IgA (e.g., IgA1 or IgA2), IgE, or IgM, and ispreferably a fragment of human or humanized IgG3 (e.g., IgG3a or IgG3b).Amino acid mutations may be introduced into the constant region of theseIgG subclasses. Amino acid mutations that can be introduced may be, forexample, those that enhance binding to Fc receptors (as described in,e.g., Proc. Natl. Acad. Sci. U.S.A. 103(11):4005-4010, 2006; MAbs 1(6):572-579, 2009; US 2010/0196362; US 2013/0108623; US 2014/0171623; US2014/0093496; and US 2014/0093959), or enhance or decrease binding toFcRn (as described in, e.g., J. Biol. Chem. 276(9):6591-6604, 2001; IntImmunol. 18(12):1759-1769, 2006; and J. Biol. Chem. 281(33):23514-23524,2006).

Two types of H chains are heterologously associated to produce abispecific Ab. The knobs-into-holes technology (as described in, e.g.,J. Immunol. Methods 248(1-2):7-15, 2001; and J. Biol. Chem. 285(27):20850-20859, 2010), the electrostatic repulsion technology (as describedin, e.g., WO 06/106905), the SEEDbody technology (as described in, e.g.,Protein Eng. Des. Sel. 23(4):195-202, 2010), and such may be used forheterologous association of two types of H chains via a CH3 domain. Anyof the antibodies described herein may be those with a modified ordeficient sugar chain. Examples of antibodies having modified sugarchains include glycosylation-engineered antibodies (as described in,e.g., WO 99/54342), antibodies with defucosylated sugar chains (asdescribed in, e.g., WO 00/61739, WO 02/31140, WO 06/067847, and WO06/067913), and antibodies having a sugar chain with bisecting GlcNAc(as described in, e.g., WO 02/79255). Known examples of methods forproducing sugar chain-deficient IgG antibodies include the method ofintroducing a mutation to asparagine at EU numbering position 297 in theheavy chain (J. Clin. Pharmacol. 50(5): 494-506, 2010), and the methodof producing IgG using E. coli (J. Immunol. Methods 263(1-2):133-147,2002; and J. Biol. Chem. 285(27):20850-20859, 2010). Furthermore,heterogeneity accompanying deletion of C-terminal lysine in IgG, andheterogeneity accompanying mispairing of disulfide bonds in the hingeregion of IgG2 can be decreased by introducing amino aciddeletions/substitutions (as described in, e.g., WO 09/041613). Any ofthe Abs or antigen-binding fragments described herein includes at leastone (e.g., one, two, three, four, five, or six) amino acids (e.g., anadded, inserted, or substituted amino acid, e.g., not within a CDR) thatare not present in a corresponding human Ab. Any of the Abs orantigen-binding fragments described herein can also have at least oneamino acid deleted (e.g., as compared to a corresponding human Ab),e.g., a deletion from the N- or C-terminus of a light or heavy chain, ora deletion of an amino acid from a constant domain (e.g., Fc domain).

SpA, or fragment thereof (e.g., at least 7, 8, 9, or 10 continuous aminoacids of SEQ ID NO: 1 (e.g., starting at amino acid position 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, or 13 of SEQ ID NO: 1), or all of SEQ IDNO: 1) can be used as an immunogen to generate Abs using standardtechniques for polyclonal and monoclonal Ab preparation. Ab fragmentscan be generated from monoclonal Abs using well-known methods in theart.

An immunogen typically is used to prepare Abs by immunizing a suitablesubject (e.g., rabbit, goat, mouse, or other mammal). An appropriateimmunogenic preparation can contain, for example, a recombinantlyexpressed or a chemically synthesized polypeptide. The preparation canfurther include an adjuvant, such as Freund's complete or incompleteadjuvant, or a similar immunostimulatory agent.

As an alternative to preparing monoclonal Ab-secreting hybridomas, amonoclonal Ab directed against a polypeptide can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an Ab phage display library) with the polypeptide of interest.Kits for generating and screening phage display libraries arecommercially available (e.g., the Pharmacia Recombinant Phage AntibodySystem, Catalog No. 27-9400-01; and the Stratagene SurfZAP* PhageDisplay Kit, Catalog No. 240612). Additionally, examples of methods andreagents particularly amenable for use in generating and screening an Abdisplay library can be found in, for example, U.S. Pat. No. 5,223,409;WO 92/18619; WO 91/17271; WO 92/2079; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; WO 90/02809; Fuchs et al., Bio/Technology9:1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas 3:81-85, 1992;Huse et al., Science 246:1275-1281, 1989; Griffiths et al., EMBO J.12:725-734, 1993.

Additional methods for isolating and sequencing a human Ab (e.g., humanIgG3) that binds specifically to a SpA epitope (e.g., an epitope locatedor defined within the polypeptide of SEQ ID NO: 1) are described in theExamples section below. Additional general methods for making Abs andantigen-binding fragments are described in U.S. Patent ApplicationPublication No. 2011/0059085.

In some embodiments, Abs or antigen-binding fragments provided hereinare human or humanized Abs (e.g., human or humanized IgG3 Abs). In someembodiments, a humanized Ab is a human Ab that has been engineered tocontain at least one complementary determining region (CDR) present in anon-human Ab (e.g., a rat, mouse, rabbit, or goat Ab). In someembodiments, a humanized Ab or fragment thereof can contain all threeCDRs of a light chain of a human or non-human Ab that specifically bindsto a SpA epitope (e.g., an epitope located or defined within thepolypeptide of SEQ ID NO: 1). In some embodiments, the humanized Ab orfragment thereof can contain all three CDRs of a heavy chain of a humanor non-human Ab that specifically binds to a SpA epitope (e.g., anepitope located or defined within the polypeptide of SEQ ID NO: 1). Insome embodiments, the humanized Ab or fragment thereof can contain allthree CDRs of a heavy chain and all three CDRs of a light chain of anon-human or human monoclonal Ab that specifically binds to a SpAepitope (e.g., an epitope located or defined within the polypeptide ofSEQ ID NO: 1).

Abs of the invention may also include multimeric forms of Abs. Forexample, Abs of the invention may take the form of Ab dimers, trimers,or higher-order multimers of monomeric immunoglobulin molecules. Dimersof whole immunoglobulin molecules or of F(ab′)₂ fragments aretetravalent, whereas dimers of Fab fragments or scFv molecules arebivalent. Individual monomers within an Ab multimer may be identical ordifferent, i.e., they may be heteromeric or homomeric Ab multimers. Forexample, individual Abs within a multimer may have the same or differentbinding specificities.

Multimerization of Abs may be accomplished through natural aggregationof Abs or through chemical or recombinant linking techniques known inthe art. For example, some percentage of purified Ab preparations (e.g.,purified IgG1 molecules) spontaneously form protein aggregatescontaining Ab homodimers and other higher-order Ab multimers.Alternatively, Ab homodimers may be formed through chemical linkagetechniques known in the art. For example, heterobifunctionalcrosslinking agents including, but not limited to, SMCC (succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidylS-acethylthio-acetate) (available, for example, from PierceBiotechnology, Inc. (Rockford, Ill.)) can be used to form Ab multimers.An exemplary protocol for the formation of Ab homodimers is given inGhetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997). Abhomodimers can be converted to Fab′₂ homodimers through digestion withpepsin. Another way to form Ab homodimers is through the use of theautophilic T15 peptide described in Zhao et al. (J. Immunol. 25:396-404,2002).

Alternatively, Abs can be made to multimerize through recombinant DNAtechniques. IgM and IgA naturally form Ab multimers through theinteraction with the mature J chain polypeptide. Non-IgA or non-IgMmolecules, such as IgG molecules, can be engineered to contain the Jchain interaction domain of IgA or IgM, thereby conferring the abilityto form higher order multimers on the non-IgA or non-IgM molecules (see,for example, Chintalacharuvu et al., Clin. Immunol. 101:21-31, 2001, andFrigerio et al., Plant Physiol. 123:1483-1494, 2000). IgA dimers arenaturally secreted into the lumen of mucosa-lined organs. This secretionis mediated through interaction of the J chain with the polymeric IgAreceptor (pIgR) on epithelial cells. If secretion of an IgA form of anAb (or of an Ab engineered to contain a J chain interaction domain) isnot desired, it can be greatly reduced by expressing the Ab molecule inassociation with a mutant J chain that does not interact well with pIgR(Johansen et al., J. Immunol., 167:5185-192, 2001). ScFv dimers can alsobe formed through recombinant techniques known in the art; an example ofthe construction of scFv dimers is given in Goel et al. (Cancer Res.60:6964-71, 2000). Ab multimers may be purified using any suitablemethod known in the art, including, but not limited to, size exclusionchromatography.

Any of the Abs or antigen-binding fragments described herein may beconjugated to a stabilizing molecule (e.g., a molecule that increasesthe half-life of the Ab or antigen-binding fragment thereof in a felineor in solution). Non-limiting examples of stabilizing molecules include:a polymer (e.g., a polyethylene glycol) or a protein (e.g., serumalbumin, such as feline serum albumin). Any of the Abs orantigen-binding fragments described herein may be conjugated to a label(e.g., a fluorophore, radioisotope, or luminescent molecule) or atherapeutic agent (e.g., a cytotoxic agent or a radioisotope). Exemplarymethods for attaching a label or therapeutic agent to an Ab aredescribed in U.S. Patent Application No. 2013/0224228. Non-limitingexamples of cytotoxic agents include agent known to induce cell death ofmicrobe (e.g., a gram positive bacterium, such as Staphylococcusaureus). Non-limiting examples of cytotoxic agents that can beconjugated to any of the Abs or antigen-binding fragments providedherein include: linezolid, erythromycin, mupirocin, ertapenem,doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin,cefalotin, cephalothin, cephalexin, ceflacor, cefamandole, cefoxitin,cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone,cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin,televancin, clindamycin, lincomycin, daptomycin, amoxicillin,ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin,flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin,penicillin G, penicillin V, piperacillin, penicillin G, temocillin,ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin,sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine,silver sulfadiazine, sulfadimethoxine, sufamethizole, sulfamethoxazole,sulfanilamide, sulfasalazine, sulfisoxazole,trimethoprim-sulfamethoxazole, sulfonamidochrysoidine, demeclocycline,doxycycline, minocycline, oxytetracycline, and tetracycline.

For example, an Ab (e.g., a human or humanized monoclonal IgG3) orantigen-binding fragment thereof (e.g., a fragment of a human orhumanized monoclonal IgG3) provided herein that specifically binds toSpA can include:

(i) a heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 2, 3,and 4, respectively, and/or a light chain comprising a CDR1, CDR2, andCDR3 of SEQ ID NOs: 7, 8, and 9, respectively;

(ii) a heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 12,13, and 14, respectively, and/or a light chain comprising a CDR1, CDR2,and CDR3 of SEQ ID NOs: 17, 18, and 19, respectively;

(iii) a heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 22,23, and 24, respectively, and/or a light chain comprising a CDR1, CDR2,and CDR3 of SEQ ID NOs: 27, 28, and 29, respectively;

(iv) a heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 32,33, and 34, respectively, and/or a light chain comprising a CDR1, CDR2,and CDR3 of SEQ ID NOs: 37, 38, and 39, respectively;

(v) a heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 42,43, and 44, respectively, and/or a light chain comprising a CDR1, CDR2,and CDR3 of SEQ ID NOs: 47, 48, and 49, respectively;

(vi) a heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 52,53, and 54, respectively, and/or a light chain comprising a CDR1, CDR2,and CDR3 of SEQ ID NOs: 57, 58, and 59, respectively;

(vii) a heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs: 62,63, and 64, respectively, and/or a light chain comprising a CDR1, CDR2,and CDR3 of SEQ ID NOs: 67, 68, and 69, respectively; or

(viii) a heavy chain comprising a CDR1, CDR2, and CDR3 of SEQ ID NOs:72, 73, and 74, respectively, and/or a light chain comprising a CDR1,CDR2, and CDR3 of SEQ ID NOs: 77, 78, and 79, respectively.

In some examples, any of the Abs provided herein has: an Ab heavy chainincluding SEQ ID NO: 6 and/or a light chain including SEQ ID NO: 11; anAb heavy chain including SEQ ID NO: 16 and/or a light chain includingSEQ ID NO: 21; an Ab heavy chain including SEQ ID NO: 26 and/or a lightchain including SEQ ID NO: 31; an Ab heavy chain including SEQ ID NO: 36and/or a light chain including SEQ ID NO: 41; an Ab heavy chainincluding SEQ ID NO: 46 and/or a light chain including SEQ ID NO: 51; anAb heavy chain including SEQ ID NO: 56 and/or a light chain includingSEQ ID NO: 61; an Ab heavy chain including SEQ ID NO: 66 and/or a lightchain including SEQ ID NO: 71; or an Ab heavy chain including SEQ ID NO:76 and/or a light chain including SEQ ID NO: 81.

In additional examples, any of the Abs (e.g., a human or humanized IgG3)or antigen-binding fragments (e.g., an antigen-binding fragment of ahuman or humanized IgG3) provided herein might bind to SpA with a K_(D)of less than 1×10⁻¹⁰ M (e.g., less than 1×10⁻¹¹ M or less than 1×10⁻¹²M) and/or be capable of displacing human Abs (e.g., one or more of IgG1,IgG2, and IgG4) bound to SpA, where the antigen or antigen-bindingfragment has a set of six CDRs has no more than one, two, three, four,five, or six total amino acid substitutions (e.g., conservative aminoacid substitutions) in the set (the entire set) of six CDRs selectedfrom the group consisting of:

(i) SEQ ID NOs: 2, 3, 4, 7, 8, and 9;

(ii) SEQ ID NOs: 12, 13, 14, 17, 18, and 19;

(iii) SEQ ID NOs: 22, 23, 24, 27, 28, and 29;

(iv) SEQ ID NOs: 32, 33, 34, 37, 38, and 39;

(v) SEQ ID NOs: 42, 43, 44, 47, 48, and 49;

(vi) SEQ ID NOs: 52, 53, 54, 57, 58, and 59;

(vii) SEQ ID NOs: 62, 63, 64, 67, 68, and 69; or

(viii) SEQ ID NOs: 72, 73, 74, 77, 78, and 79.

For example, an Ab (e.g., a human or humanized IgG3) or anantigen-binding fragment (e.g., an antigen-binding fragment of a humanor humanized IgG3) provided herein can include a set of six CDRs thathas no more than one, two, three, or four total amino acid substitutionsin the set (the entire set) of six CDRs of SEQ ID NOs: 2, 3, 4, 7, 8,and 9. For example, an Ab (e.g., a human or humanized IgG3) orantigen-binding fragment (e.g., an antigen binding fragment of a humanor humanized IgG3) provided herein can comprise or consist of:

(i) a set of six CDRs of SEQ ID NOs: 2, 3, 4, 7, 8, and 9;

(ii) a set of six CDRs of SEQ ID NOs: 12, 13, 14, 17, 18, and 19;

(iii) a set of six CDRs of SEQ ID NOs: 22, 23, 24, 27, 28, and 29;

(iv) a set of six CDRs of SEQ ID NOs: 32, 33, 34, 37, 38, and 39;

(v) a set of six CDRs of SEQ ID NOs: 42, 43, 44, 47, 48, and 49;

(vi) a set of six CDRs of SEQ ID NOs: 52, 53, 54, 57, 58, and 59;

(vii) a set of six CDRs of SEQ ID NOs: 62, 63, 64, 67, 68, and 69; or

(viii) a set of six CDRs of SEQ ID NOs: 72, 73, 74, 77, 78, and 79.

In additional examples, an Ab (e.g., a human or humanized monoclonalIgG3) or antigen-binding fragment (e.g., an antigen-binding fragment ofa human or humanized IgG3) provided herein that specifically binds toSpA includes a variable domain selected from the group of: (i) avariable domain comprising or consisting of SEQ ID NO: 5; (ii) avariable domain comprising or consisting of SEQ ID NO: 10; (iii) avariable domain comprising or consisting of SEQ ID NO: 15; (iv) avariable domain comprising or consisting of SEQ ID NO: 20; (v) avariable domain comprising or consisting of SEQ ID NO: 25; (vi) avariable domain comprising or consisting of SEQ ID NO: 30; (vii) avariable domain comprising or consisting of SEQ ID NO: 35; (viii) avariable domain comprising or consisting of SEQ ID NO: 40; (ix) avariable domain comprising or consisting of SEQ ID NO: 45; (x) avariable domain comprising or consisting of SEQ ID NO: 50; (xi) avariable domain comprising or consisting of SEQ ID NO: 55; (xii) avariable domain comprising or consisting of SEQ ID NO: 60; (xiii) avariable domain comprising or consisting of SEQ ID NO: 65; (xiv) avariable domain comprising or consisting of SEQ ID NO: 70; (xv) avariable domain comprising or consisting of SEQ ID NO: 75; or (xvi) avariable domain comprising or consisting of SEQ ID NO: 80. For example,an Ab (e.g., a human or humanized monoclonal IgG3) or antigen-bindingfragment (e.g., an antigen-binding fragment of a human or humanizedIgG3) can include (i) a variable domain comprising or consisting of SEQID NO: 5 and/or a variable domain comprising or consisting of SEQ ID NO:10; (ii) a variable domain comprising or consisting of SEQ ID NO:15and/or a variable domain comprising or consisting of SEQ ID NO: 20;(iii) a variable domain comprising or consisting of SEQ ID NO: 25 and/ora variable domain comprising or consisting of SEQ ID NO: 30; (iv) avariable domain comprising or consisting of SEQ ID NO: 35 and/or avariable domain comprising or consisting of SEQ ID NO: 40; (v) avariable domain comprising or consisting of SEQ ID NO: 45 and/or avariable domain comprising or consisting of SEQ ID NO: 50; (vi) avariable domain comprising or consisting of SEQ ID NO: 55 and/or avariable domain comprising or consisting of SEQ ID NO: 60; (vii) avariable domain comprising or consisting of SEQ ID NO: 65 and/or avariable domain comprising or consisting of SEQ ID NO: 70; or a variabledomain comprising or consisting of SEQ ID NO: 75 and/or a variabledomain comprising or consisting of SEQ ID NO: 80.

Some embodiments of any of the Abs (e.g., human or humanized monoclonalIgG3) or antigen-binding fragments (e.g., an antigen-binding fragment ofa human or humanized IgG3) described herein have one or more (e.g., one,two, three, or four) of the following activities: specifically bind toSpA in a strain of MRSA; specifically bind to an epitope defined by SEQID NO: 1; bind to SpA with a K_(D) of less than 1×10⁻¹⁰ M (e.g., lessthan 1×10⁻¹¹ M or less than 1×10⁻¹²); and displace human Abs bound toSpA in the cell wall of a Staphylococcus aureus bacterium (e.g., a MRSAbacterium).

Pharmaceutical Compositions

Provided herein are pharmaceutical compositions containing at least onepharmaceutically acceptable carrier (e.g., a non-naturalpharmaceutically acceptable carrier) and at least one (e.g., two, three,or four) of any of the Abs or antigen-binding fragments provided herein.Non-limiting examples of pharmaceutically acceptable carriers includesterilized water, physiological saline, stabilizers, excipients,antioxidants (e.g., ascorbic acid), buffers (e.g., phosphate, citrate,histidine, and other organic acids), antiseptics, surfactants (e.g., PEGand Tween), chelating agents (e.g., EDTA or EGTA), and binders.Additional examples of pharmaceutically acceptable carriers also includelow-molecular-weight polypeptides, proteins (e.g., serum albumin andgelatin), amino acids (e.g., glycine, glutamine, asparagine, glutamicacid, asparagic acid, methionine, arginine, and lysine), sugars andcarbohydrates (e.g., polysaccharides and monosaccharides), and sugaralcohols (e.g., mannitol and sorbitol). When preparing an aqueoussolution for injection, physiological saline and isotonic solutionscomprising glucose and other adjuvants such as D-sorbitol, D-mannose,D-mannitol, and sodium chloride may be used, and if necessary, incombination with appropriate solubilizers, such as alcohol (e.g.,ethanol), polyalcohols (e.g., propylene glycol and PEG), and nonionicsurfactants (e.g., polysorbate 80, polysorbate 20, poloxamer 188, andHCO-50). By mixing hyaluronidase into the formulation, a larger fluidvolume can be administered subcutaneously (see, e.g., Expert. Opin.Drug. Deliv. 4(4): 427-440, 2007).

The Abs and antigen-binding fragments provided herein may, e.g., beencapsulated in microcapsules (e.g., those made ofhydroxymethylcellulose, gelatin, and poly(methylmetacrylate)), orincorporated as components of colloidal drug delivery systems (e.g.,liposomes, albumin microspheres, microemulsion, nanoparticles, andnanocapsules) (see, for example, “Remington's Pharmaceutical Science16th edition”, Oslo Ed. (1980)). Methods for preparing thepharmaceutical compositions as controlled-release pharmaceutical agentsare also well-known, and such methods may be applied to the Abs andantigen-binding fragments of the present invention (see, e.g., Langer etal., J. Biomed. Mater. Res. 15: 267-277, 1981; Langer, Chemtech. 12:98-105, 1982; U.S. Pat. No. 3,773,919; European Patent ApplicationPublication No. EP 58,481; Sidman et al., Biopolymers 22: 547-556, 1983;and EP 133,988).

The pharmaceutical compositions provided herein can be formulated forintravenous, intaarterial, intradermally, subcutaneous, intramuscular,intraperitoneal, or oral administration.

The dose of a pharmaceutical composition of the present invention may beappropriately determined by considering the dosage form, method ofadministration, patient age and body weight, symptoms of the patient,severity of the SA infection, or level of risk of SA infection.Generally, the daily dose for an adult can be, e.g., between 0.1 mg to10,000 mg at once or in several portions. The dose can be, e.g., 0.2 to10,000 mg/day (e.g., 1-10 g/day, 2-8 g/day, 1-5 g/day, 0.5 to 2.5 g/day,0.5 to 500 mg/day, 1 to 300 mg/day, 3 to 100 mg/day, or 5 to 50 mg/day).These doses may vary, depending on the patient body weight and age, andthe method of administration; however, selection of suitable dosage iswell within the purview of those skilled in the art. Similarly, thedosing period may be appropriately determined depending on thetherapeutic progress.

Any of the pharmaceutical compositions provided herein can furtherinclude one or more additional antimicrobial agents. Non-limitingexamples of such antimicrobial agents include: linezolid, erythromycin,mupirocin, ertapenem, doripenem, imipenem, cilastatin, meropenem,cefadroxil, cefazolin, cefalotin, cefalothin, cephalexin, ceflacor,cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir,cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, ceftaroline fosamil, ceftobiprole,teicoplanin, vancomycin, televancin, clindamycin, lincomycin,daptomycin, amoxicillin, ampicillin, azlocillin, carbenicillin,cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin,nafcillin, oxacillin, penicillin G, penicillin V, piperacillin,penicillin G, temocillin, ticarcillin, bacitracin, colistin, polymyxinB, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin,lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin,trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide,sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine,sufamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine,sulfisoxazole, trimethoprim-sulfamethoxazole, sulfonamidochrysoidine,demeclocycline, doxycycline, minocycline, oxytetracycline, andtetracycline.

Methods of Treating a Subject Having a S. aureus Infection or Reducingthe Risk of Developing a S. Aureus Infection in a Subject

Also provided are methods of treating a subject having a SA infection(e.g., MRSA infection, SA bacteremia, SA skin infection, SA mastitis, SAcellulitis or folliculitis, or SA-involved wound infections, abscesses,osteomyelitis, endocarditis, pneumonia, septic shock, food poisoning, ortoxic shock syndrome) that include administering to a subject (e.g., ahuman being or another mammal such as a bovine, ovine, canine, feline,equine, hircine, leporine, porcine, or avian) in need thereof atherapeutically effective amount of at least one of any of thepharmaceutical compositions provided herein or at least one of any ofthe Abs or antigen-binding fragments provided herein. In some examples,the subject has been diagnosed or identified as having a SA infection(e.g., a MRSA infection). Some embodiments further include (prior to theadministering step) a step of diagnosing, identifying, or selectingsubject having or as having a SA infection (e.g., a MRSA or VRSAinfection). In some examples, the SA infection is a nosocomialinfection. In some examples, the subject has previously been treatedwith an antibacterial treatment and the prior treatment wasunsuccessful.

Also provided are methods of reducing a subject's risk of developing aSA infection (e.g., a MRSA infection) that include administering to thesubject an effective amount of at least one of any of the pharmaceuticalcompositions provided herein or at least one of any of the Abs orantigen-binding fragments provided herein. In some embodiments, the SAinfection is a nosocomial infection. Some embodiments further includeprior to administering selecting or identifying a subject as having anincreased risk of developing a SA infection (e.g., a MRSA infection).For example, the subject can be a medical professional (e.g., aphysician, a nurse, a laboratory technician, or a physician's assistant)(e.g., a medical professional in physical contact with a subject havinga SA infection (e.g., a MRSA infection)). A subject in these methods canalso be a subject admitted to a hospital or inpatient treatment (e.g., anursing home) that contains (has admitted) at least one other subjecthaving a SA infection (e.g., a MRSA infection). The subject may be ahospitalized patient such as one in the intensive care unit, animmunocompromised patient, and a patient who has undergone or willundergo a surgical procedure (e.g, cardiac surgery).

In any of the methods provided herein, the subject can be a male or afemale. For example, the subject can an infant, a toddler, anadolescent, a teenager, or an adult (e.g., at least 18 years old, atleast 20 years old, at least 25 years old, at least 30 years old, atleast 35 years old, at least 40 years old, at least 45 years old, atleast 50 years old, at least 55 years old, at least 60 years old, atleast 65 years old, at least 70 years old, at least 75 years old, atleast 80 years old, at least 85 years old, at least 90 years old, atleast 95 years old, or at least 100 years old). In some examples, thesubject has a suppressed or weakened immune system (e.g., humoral orcellular immune system).

In some examples, the at least one pharmaceutical composition providedherein or at least one Ab or antigen-binding fragment provided herein isadministered by intravenous, intaarterial, intradermally, subcutaneous,intramuscular, intraperitoneal, or oral administration. For example, inmethods of reducing the risk of developing a SA infection, the subjectis administered at least one of the pharmaceutical compositions providedherein or at least one of the Abs or antigen-binding fragments providedherein prior to or shortly after coming into physical contact with asubject identified, diagnosed, having, or suspected of having SAinfection (e.g., a MRSA infection).

In any of the methods described herein, the subject is administered atleast one (e.g., two, three, four, five, six, seven, eight, nine, orten) dose(s) of any of the pharmaceutical compositions provided hereinor at least one (e.g., two, three, four, five, six, seven, eight, nine,or ten) dose(s) of any of the Abs or antigen-binding fragments providedherein. A subject can be administered two of more doses of any of thepharmaceutical compositions or at least two doses of any of the Abs orantigen-binding fragments provided herein at a frequency of at least onedose every month (e.g., at least two doses every month, at least threedoses every month, at least four doses every month, at least one dose aweek, at least two doses a week, at least three doses a week, at leastfour doses a week, at least five doses a week, at least one dose a day,at least two doses a day, or at least three doses a day).

Some embodiments further include co-administering to a subject and Abdescribed herein and one or more additional antimicrobial agents.Non-limiting examples of such antimicrobial agents include: linezolid,erythromycin, mupirocin, ertapenem, doripenem, imipenem, cilastatin,meropenem, cefadroxil, cefazolin, cefalotin, cefalothin, cephalexin,ceflacor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime,cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime,ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, ceftaroline fosamil,ceftobiprole, teicoplanin, vancomycin, televancin, clindamycin,lincomycin, daptomycin, amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin,methicillin, nafcillin, oxacillin, penicillin G, penicillin V,piperacillin, penicillin G, temocillin, ticarcillin, bacitracin,colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin,gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid,norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin,temafloxacin, mafenide, sulfacetamide, sulfadiazine, silversulfadiazine, sulfadimethoxine, sufamethizole, sulfamethoxazole,sulfanilamide, sulfasalazine, sulfisoxazole,trimethoprim-sulfamethoxazole, sulfonamidochrysoidine, demeclocycline,doxycycline, minocycline, oxytetracycline, and tetracycline. Additionalexamples of therapeutic agents that can be included in any of thepharmaceutical compositions provided herein are one or more Absdescribed in U.S. Patent Application Publication No. 2011/0059085.

Kits

Also provided herein are kits containing at least one (e.g., two, three,four, or five) of any of the Abs or antigen-binding fragments providedherein. In some examples, the kits can contain a recombinant SpA or apeptide comprising or consisting of SEQ ID NO: 1 or an antigenicfragment of SEQ ID NO: 1 (e.g., at least 7 contiguous amino acids of SEQID NO: 1 (e.g., starting at amino acids position 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or 13 of SEQ ID NO: 1)). In some examples, the at leastone Ab or antigen-binding fragment is attached to a solid substrate(e.g., a well, a chip, a film, a bead, or a chromatography resin). Suchkits can include commercial packaging and/or printed information aboutthe Abs and methods of their use.

EXAMPLES Example 1 Generation of Human Abs that Specifically Bind to SpAand Displace Human IgG Immunoglobulins Bound to SpA Via their Fc Region

Human IgG3 Abs that bind to a SpA epitope were generated as describedbelow. Five synthesized peptides covering the IgG-binding and Xr repeatsequences in SpA were used to screen for anti-peptide Abs in the bloodof 311 healthy adult volunteers. The five synthesized peptides from SpAused for screening had the sequences indicated as: SEQ ID NOs: 82, 83,84, 85, and 1 (peptides 1, 2, 3, 4, and 5, respectively). About 4% ofthe healthy subjects had greater than 10-fold higher levels ofanti-peptide (anti-SpA) Abs over background (hereafter called “positivedonors”) as determined using an enzyme-linked immunosorbent assay(ELISA). Plasma from these positive donors was obtained and used toisolate true human Abs that bind specifically to a peptide covering theIgG-binding and Xr repeat sequences of SpA using the methods describedin U.S. Patent Application Publication No. 2013/0018173. In sum, Abs ofinterest were isolated using antigen affinity chromatography, and denovo sequenced using mass spectrometry. In parallel, the Abs wereisotyped using a human isotyping kit.

One of the isolated Abs was identified as being in the VH3 subfamily andhaving an IgG2 heavy chain and VK1 light chain. B-cells were isolatedfrom the donor blood using a kit obtained from STEMCELL Technologies,Inc. Their RNA was extracted using a Trizol extraction protocol, andcDNA was generated using SuperScript III. Leader-specific primers wereused to amplify the corresponding heavy and light chains of the Ab and a“directed” ScFv library was generated. The library was panned againstwildtype SpA antigen for 7 rounds. The clones were screened using directand sandwich ELISA with wildtype SpA. The selected clones weresequenced, and the heavy and light chains were cloned into vectors withan IgG3 constant (Fc) region (one that lacks the SpA recognition site inthe Fab regions). The vectors were transfected into CHO cell lines, andhigh producing clones were picked. The purified Abs were tested foranti-SpA activity. The clones were scaled up for large-scale production,and the produced Abs were purified and used for further analyses.Examples of eight such Abs are described below:

PA8-G3 Ab

Heavy chain variable domain of SEQ ID NO: 5.

Heavy chain CDRs 1, 2, and 3 of SEQ ID NOs: 2, 3, and 4, respectively.

Heavy chain of SEQ ID NO: 6.

Light chain variable domain of SEQ ID NO: 10.

Light chain CDRs 1, 2, and 3 of SEQ ID NO: 7, 8, and 9, respectively.

Light chain of SEQ ID NO: 11.

PA4-G3 Ab

Heavy chain variable domain of SEQ ID NO: 15.

Heavy chain CDRs 1, 2, and 3 of SEQ ID NOs: 12, 13, and 14,respectively.

Heavy chain of SEQ ID NO: 16.

Light chain variable domain of SEQ ID NO: 20.

Light chain CDRs 1, 2, and 3 of SEQ ID NOs: 17, 18, and 19,respectively.

Light chain of SEQ ID NO: 21.

PA7.2-G3 Ab

Heavy chain variable domain of SEQ ID NO: 25.

Heavy chain CDRs 1, 2, and 3 of SEQ ID NOs: 22, 23, and 24,respectively.

Heavy chain of SEQ ID NO: 26.

Light chain variable domain of SEQ ID NO: 30.

Light chain CDRs 1, 2, and 3 of SEQ ID NOs: 27, 28, and 29,respectively.

Light chain of SEQ ID NO: 31.

PA15-G3 Ab

Heavy chain variable domain of SEQ ID NO: 35.

Heavy chain CDRs 1, 2, and 3 of SEQ ID NOs: 32, 33, and 34,respectively.

Heavy chain of SEQ ID NO: 36.

Light chain variable domain of SEQ ID NO: 40.

Light chain CDRs 1, 2, and 3 of SEQ ID NOs: 37, 38, and 39,respectively.

Light chain of SEQ ID NO: 41.

PA21-G3 Ab

Heavy chain variable domain of SEQ ID NO: 45.

Heavy chain CDRs 1, 2, and 3 of SEQ ID NOs: 42, 43, and 44,respectively.

Heavy chain of SEQ ID NO: 46.

Light chain variable domain of SEQ ID NO: 50.

Light chain CDRs 1, 2, and 3 of SEQ ID NOs: 47, 48, and 49,respectively.

Light chain of SEQ ID NO: 51.

PA27-G3 Ab

Heavy chain variable domain of SEQ ID NO: 55.

Heavy chain CDRs 1, 2, and 3 of SEQ ID NOs: 52, 53, and 54,respectively.

Heavy chain of SEQ ID NO: 56.

Light chain variable domain of SEQ ID NO: 60.

Light chain CDRs 1, 2, and 3 of SEQ ID NOs: 57, 58, and 59,respectively.

Light chain of SEQ ID NO: 61.

PA32-G3 Ab

Heavy chain variable domain of SEQ ID NO: 65.

Heavy chain CDRs 1, 2, and 3 of SEQ ID NOs: 62, 63, and 64,respectively.

Heavy chain of SEQ ID NO: 66.

Light chain variable domain of SEQ ID NO: 70.

Light chain CDRs 1, 2, and 3 of SEQ ID NO: 67, 68, and 69, respectively.

Light chain of SEQ ID NO: 71.

PA37-G3 Ab

Heavy chain variable domain of SEQ ID NO: 75.

Heavy chain CDRs 1, 2, and 3 of SEQ ID NOs: 72, 73, and 74,respectively.

Heavy chain of SEQ ID NO: 76.

Light chain variable domain of SEQ ID NO: 80.

Light chain CDRs 1, 2, and 3 of SEQ ID NOs: 77, 78, and 79,respectively.

Light chain of SEQ ID NO: 81.

A set of experiments was performed to determine whether the PA8-G3 Abwould be capable of binding to SpA on the cell wall of SA. In theseexperiments, SA stains ATCC #25923 or clinical isolate 00X wereincubated either with (i) biotinylated PA8-G3, and then streptavidin-APCto fluorescently quantify the amount of biotin-PA-G3 bound on the SAsurface (FIG. 2) or (ii) purified unlabeled PA8-G3 Ab, followed bybiotinylated recombinant Fcγ receptor 1, and then streptavidin-APC tofluorescently quantify the amount of PA8-G3 bound to the SA surface thatwould lead to phagocytosis (i.e., have free Fc regions available to bindthe recombinant Fcγ receptor 1) (FIG. 3). An anti-interleukin-1a Ab(MABp1) was used as a negative control in these experiments. The data inFIG. 2 show that PA8-G3 binds to SpA in the cell wall of SA and the datain FIG. 3 indicate that the bound PA8-G3 Ab had its Fc regions availableto interact with FcR suggesting that the Ab would able to mediateopsinophagocytosis of SA in human subjects (as opposed to having its Fcregions bound to SpA and not able to engage FcRs and therefore mediateopsinophagocytosis of the bacteria).

A further set of experiments was performed to test whether binding ofPA8-G3 Ab to the surface of SA would be recognized by the Fc γ receptorson phagocytes. In these experiments, two different strains ofpH-rodo-green labeled S. aureus (clinical isolate 00X or ATCC #25923)were incubated with either unlabeled PA8-G3 Ab or a control Ab (MABp1),and then incubated with differentiated HL-60 cells. The resultingfluorescence of the HL-60 cells was determined usingfluorescence-assisted cell sorting (FACS). The data show that PA8-G3binds to the cell wall of both SA strains and mediates phagocytosisthrough the Fcγ receptors on the surface of HL-60 cells (FIG. 4). Thesuccessful phagocytosis by differentiated HL-60 cells of S. aureus boundto PA8-G3 was also evident from fluorescence microscopy experiments.

Surface plasmon resonance was used to determining the binding kineticsof PA8-G3 to SpA. In these experiments, PA8-G3 Ab was immobilized usinganti-human capture sensor and commercial wildtype SpA. These data showthat PA8-G3 has a K_(D) of 5.38 pM. This affinity is approximately1000-fold higher than the nanomolar affinity of human serum IgG1, IgG2,and IgG4 to SpA.

An additional set of experiments was performed to determine whetherPA8-G3 Ab would be able to successfully compete for binding to SpA withhuman IgG bound to SpA through their Fc receptor. In these experiments,two different S. aureus strains were pre-incubated with human sera(which contains a high concentration of Igs which bind SpA via their Fcregions) for 15 minutes prior to incubation with biotinylated PA8-G3 Abor biotinylated MABp1-IgG3 Ab (isotype-matched negative control), thentreated with streptavidin APC, and then fluorescence was determined byflow cytometry. The data show that PA8-G3 Ab was able bind SpA havinghuman IgG Abs bound to SpA by their Fc domain (FIG. 5).

In another set of experiments, PA8-G3 antibody was shown to compete withMABp1-IgG1 (which binds SpA via its Fc region) binding on SpA-coatedbeads. Pre-incubating the SpA beads wth PA8-G3 reduced later addedMABp1-IgG1 binding by 80.3%. Conversely, with SpA beads pre-incubatedwith MABp1-IgG1, later added PA8-G3 bound greater than >30% of the SpAbeads surfaces within 15 minutes, whereas later added MABp1-IgG3(isotype-matched negative control having the Fab of MABp1 and a humanIgG3 Fc) did not significantly bind to SpA beads pre-incubated withMABp1-IgG1.

An additional set of experiments was performed to test the ability ofadditional anti-SpA Abs to promote phagocytosis of SA by differentiatedHL-60 cells. In these experiments, differentiated HL-60 cells wereco-incubated with pH-rodo-green labeled S. aureus and one of thefollowing Abs: PA7.2-G3, PA4-G3, PA8-G3, PA15-G3, PA21-G3, PA27-G3,PA32-G3, PA37-G3, or MABp1. MABp1 was used as a negative control inthese experiments. The data show that all of the tested anti-SpA Abswere able to promote opsinization and phagocytosis of S. aureus bydifferentiated HL-60 cells (FIG. 6).

Additional Bio-Layer Interferometry (using done using a ForteBio OctetRed 96 instrument) experiments were performed to determine the K_(D) ofseven additional anti-SpA Abs (performed using 20 nM antigen). Theresulting data showed that PA7.2-G3 has a K_(D) of less than 1×10⁻¹² M,PA4-G3 has a K_(D) of 5.38×10⁻¹² M, PA15-G3 has a K_(D) of less than1×10⁻¹² M, PA21-G3 has a K_(D) of less than 1×10⁻¹² M, PA27-G3 has aK_(D) of less than 1×10⁻¹² M, PA32-G3 has a K_(D) of less than 1×10⁻¹²M, and PA37-G3 has a K_(D) of less than 1×10⁻¹² M.

In sum, the data show that the Abs provided herein can bind with veryhigh affinity to SpA in the cell wall of SA, promote phagocytosis byimmune cells, and are capable of doing so in the presence of human IgGsbound to SpA by their Fc domain.

Example 2 In Vivo Survival Study of Monoclonal Antibody PA8 in MiceBacteremia/Sepsis Model

Survival of mice from S. aureus bacteremica was examined usingprophylactic doses of PA8 (the monoclonal antibody termed PA8-G3described in Example 1).

Female Balb/C mice (6-8 weeks of age) were purchased from Charles RiverLaboratory, NIH, Maryland. Upon arrival, the mice were examined, grouphoused (10/cage) in cages with absorbent bedding. All mice were placedunder the required husbandry standards found in the NIH Guide for theCare and Use of Laboratory Animals.

The protective efficacy of PA8 was investigated in the SA sepsis modelinduced by intravenous injections (i.v.) of 2×10⁷ CFUs of MRSA strainNR-46223. Mice were treated intravenously with PA8 at specific doses (5mg or 10 mg) 3 h prior to MRSA infection or two doses of 5 mg each atday 0 and 3. Control mice were treated with formulation buffer only. Themice were followed for 10 days (twice per day) at which point allremaining mice were sacrificed.

Three hours after the PA8/formulation buffer (0.1 ml) i.v.administration, the mice were challenged with a single intravenous (IV)injection of S. aureus strain NR-46223 (2×10⁷ CFU in 0.1 ml). One set ofmice was given two doses of 5 mg each at day 0 and 3. Significantdifferences in the relative survival times between treatment groups weredetected. Referring to FIG. 7A-D, passive administration of single doseof 5 mg (A) or 10 mg (B), or two doses of 5 mg at day 0 & day 3 (C), ofmAb PA8 (intravenously) enhances the survival of BALB/c micesignificantly higher than formulation buffer treatment in dose dependentmanner (10 mice per group) with Staphylococcus aureus sepsis (induced byintravenous injection of 2×10⁷ colony-forming units ofmethicillin-resistant S. aureus strain NR-46223). Section (D) shows thesurvival using all different treatment in one graph. Fifty percent (5/10) of the mice survived that received 5 mg of Mab PA8 (p=0.016),sixty percent that received two doses of 5 mg each (p=0.09), and seventypercent ( 7/10) that received 10 mg of mAb PA8 (p=0.003) compared to 10%( 1/10) of mice that received formulation buffer ( 1/10) survived thebacterial challenge with S. aureus NR-46223. Statistical analysis of theanimal data was conducted using Kaplan-Meier Survival Analysis with aMantel-Cox (logrank) test. These results clearly indicate that PA8provides a significant level of protection against lethal infection withS. aureus MRSA strain.

Example 3

Female Balb/C mice (10 per group) from Charles River Laboratory wereinjected with 0.5 mg of vancomycin via intraperitoneal route, along withdifferent sub-optimal doses of PA8-G3 (0 mg, 2.5 mg and 5 mg viaintravenous route) two hours prior to infection with MRSA (NR 46223 at3×10⁷ CFU i.v.). The mice were observed for 14 days. Referring to FIGS.8A-C, at day 14, only 10% of the PBS treated mice survived, 30% of thevancomycin treated mice survived. However, when 2.5 mg of PA8-G3 wasinjected along with vancomycin treatment, then 60% of the animalssurvived (p=0.027), and when 5 mg of PA8-G3 was injected withvancomycin, then 60% of the animals survived and those mice that diedlived longer than the lower dosage (p=0.016). This data indicates thatsub-efficacious doses of PA8-G3 can rescue animals from SA mediatedbacteremia, when co-treated with sub-optimal dose of vancomycin.Statistical analysis of the animal data was conducted using Kaplan-MeierSurvival Analysis with a Mantel-Cox (logrank) test.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a purified monoclonal antibodycomprising a heavy chain variable domain comprising a CDR1, CDR2, andCDR3 of SEQ ID NO: 2, 3, and 4, respectively, and a light chain variabledomain comprising a CDR1, CDR2, and CDR3 of SEQ ID NO: 7, 8, and 9,respectively, wherein the monoclonal antibody specifically bindsStaphylococcus aureus protein A (SpA) with a K_(D) of less than 1×10⁻¹⁰M via its Fab region paratope, wherein the monoclonal antibody is ableto displace human IgG immunoglobulins bound to SpA on Staphylococcusaureus bacteria via their Fc regions.
 2. The pharmaceutical compositionof claim 1, wherein the monoclonal antibody is able to mediateopsonization of SpA-expressing Staphylococcus aureus bacteria in thepresence of at least 1 mg/ml of IgG immunoglobulins which bind SpA viatheir Fc regions.
 3. The pharmaceutical composition of claim 1, whereinthe monoclonal antibody is a human or humanized IgG3 monoclonalantibody.
 4. The pharmaceutical composition of claim 1, wherein themonoclonal antibody specifically binds to the amino acid sequence SEQ IDNO:
 1. 5. The pharmaceutical composition of claim 1, wherein the heavychain variable domain comprises SEQ ID NO: 5 and the light chainvariable domain comprises SEQ ID NO:
 10. 6. The pharmaceuticalcomposition of claim 5, wherein the monoclonal antibody is able tomediate opsonization of SpA-expressing Staphylococcus aureus bacteria inthe presence of at least 1 mg/ml of IgG immunoglobulins which bind SpAvia their Fc regions.
 7. The pharmaceutical composition of claim 5,wherein the monoclonal antibody is a human or humanized IgG3 monoclonalantibody.
 8. The pharmaceutical composition of claim 5, wherein themonoclonal antibody specifically binds to the amino acid sequence SEQ IDNO: 1.